Test media and quantitative or qualitative method for identification and differentiation of biological materials in a test sample

ABSTRACT

A test medium and method for detecting, quantifying, identifying and differentiating up to four (4) separate biological materials in a test sample. A test medium is disclosed which allows quantifying and differentiating under ambient light aggregates of biological entities producing specific enzymes, which might include general coliforms,  E. coli, Aeromonas , and  Salmonella  in a single test medium. A new class of nonchromogenic substrates is disclosed which produce a substantially black, non-diffusible precipitate. This precipitate is not subject to interference from other chromogenic substrates present in the test medium. In one embodiment, the substrates are selected such that  E. coli  colonies present in the test medium show as substantially black, general coliforms colonies show in the test medium as a blue-violet color,  Aeromonas  colonies present in the test medium show as a generally red-pink color, and  Salmonella  colonies show as a generally teal-green color. Other microorganisms and color possibilities for detection and quantification thereof are also disclosed. An inhibitor and method for making a test medium incorporating the inhibitor are disclosed.

CLAIM OF PRIORITY

[0001] This Application is a continuation-in-part of U.S. applicationSer. No. 10/040,792 filed on Jan. 7, 2002, which is a continuation ofU.S. application Ser. No. 09/357,606 filed on Jul. 20, 1999 and issuedas U.S. Pat. No. 6,350,588, all of which are incorporated by referenceas if fully rewritten herein.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to a test medium and method for thedetection, quantification, identification and/or differentiation ofbiological materials in a sample which may contain a plurality ofdifferent biological materials.

[0003] Bacteria are the causative factor in many diseases of humans,higher animals and plants, and are commonly transmitted by carriers suchas water, beverages, food and other organisms. The testing of thesepotential carriers of bacteria is of critical importance and generallyrelies on “indicator organisms.” Borrego et al., Microbiol. Sem.13:413-426, (1998). For example, Escherichia coli (E. coli) is a gramnegative member of the family Enterobacteriaceae which is part of thenormal intestinal flora of warm blooded animals, and its presenceindicates fecal contamination (e.g., raw sewage). Even though moststrains of E. coli are not the actual cause of disease, their presenceis a strong indication of the possible presence of pathogens associatedwith intestinal disease, such as cholera, dysentery, and hepatitis,among others. Consequently, E. coli has become a prime indicatororganism for fecal contamination, and as a result, any method whichdifferentiates and identifies E. coli from other bacteria is veryuseful.

[0004] Others members of the family Enterobacteriaceae, commonlyreferred to as “general coliforms,” especially the genera Citrobacter,Enterobacter and Klebsiella, are also considered to be significantindicator organisms for the quality of water, beverages and foods.Therefore, tests to identify and differentiate general coliforms from E.coli are also very useful. Also, various species of the genus Aeromonashave been shown to not only be potential pathogens, but to have acorrelation to other indicator organisms (Pettibone et al., J. Appl.Microbiol. 85:723-730 (1998)). Current test methods to identify,separate and enumerate Aeromonas spp. from the very similarEnterobacteriaceae have been lacking and most of the current methodsutilizing enzyme substrates do not separate Aeromonas spp. fromEnterobacteriaceae due to their almost identical biochemical profiles.Any method that depends upon the identification of general coliforms bymeans of a β-galactosidase substrate either does not differentiateAeromonas spp. from general coliforms or eliminates Aeromonas from thesample by the use of specific inhibitors (antibiotic such ascefsulodin). Brenner et al., Appl. Envir. Microbio. 59:3534-44 (1993).They do not differentiate, identify and enumerate Aeromonas along withE. coli and general coliforms. Landre et al., Letters Appl. Microbiol.26:352-354(1998). Improved test methods to effectively identify,separate and enumerate such bacterial types are needed, and there is acontinuing search for faster, more accurate, easier to use and moreversatile test methods and apparatus in this area.

[0005] Numerous test methods have been utilized to determine, identifyand enumerate one or more indicator organisms. Some of these testmethods only indicate the presence or absence of the microorganism,while others also attempt to quantify one or more of the particularorganisms in the test sample. For example, a qualitative test referredto as the Presence/Absence (or P/A) test, may be utilized to determinethe presence or absence of coliforms and E. coli in a test sample. Atest medium including the β-galactosidase substrateO-nitrophenyl-β-D-galactopyranoside (ONPG), and the β-glucuronidasesubstrate 4-methyl-umbrelliferyl-β-D-glucuronide (MUG), is inoculatedwith the test sample. To differentiate the general coliforms from E.coli, this test relies on the fact that generally all coliforms produceβ-galactosidase, whereas only E. coli also produces β-glucuronidase inaddition to β- galactosidase. If any coliforms are present (including E.coli), the broth medium turns a yellow color due to the activity of thegalactosidase enzyme on the ONPG material, causing the release of adiffusible yellow pigment. If E. coli is present, the broth medium willdemonstrate a blue fluorescence when irradiated with ultraviolet rays,due to the breakdown of the MUG reagent with the release of thefluorogenic dye caused by the production of the glucuronidase enzyme.These reactions are very specific, and allow the presence of bothcoliforms in general, as well as E. coli to be identified in a singlesample. A disadvantage of this test is that it is not directlyquantitative for either bacterial type, since both reagents producediffusible pigments. A second disadvantage is that there may a falsepositive coliform reaction if Aeromonas spp. are present in the testsample. This has been shown to be possible even when there areinhibitors present to supposedly prevent this from occurring (Landre etal., Letters Appl. Microbiol. 26:352-354 (1998)). The test also requiresspecific equipment for producing the ultraviolet rays. Further, thistest may only be used to detect coliforms and E. coli. Other importantmicroorganisms, such as the strain E. coli 0157 which is glucuronidasenegative, are not detected, nor are othernon-galactosidase-glucuronidase producing microorganisms.

[0006] The Violet Red Bile Agar (VRBA) method has been used to determinethe quantity of both coliform and E. coli in a test sample. The testmedium used in this method includes bile salts (to inhibitnon-coliforms), lactose and the pH indicator neutral red. As coliforms(including E. coli) grow in the medium, the lactose is fermented withacid production, and the neutral red in the area of the bacterial colonybecomes a brick red color. The results of this test are not always easyto interpret, and in order to determine the presence of E. coli,confirming follow-up tests, such as brilliant green lactose brothfermentation, growth in EC broth at 44.5° C. and streaking on EosinMethylene Blue Agar (EMBA), must be performed.

[0007] The Membrane Filter (MF) method utilizes micropore filtersthrough which samples are passed so that the bacteria are retained onthe surface of the filter. This method is used most often when bacterialpopulations are very small, and a large sample is needed to get adequatenumbers. The filter is then placed on the surface of a chosen medium,incubated, and the bacterial colonies growing on the membrane filtersurface are counted and evaluated. This method is widely used andprovides good results when combined with proper reagents and media. Adisadvantage of this method is that it is expensive and time-consuming.It also does not work well with solid samples, or samples with highparticulate counts. The MF method can be used in conjunction with theinventive method described in this application.

[0008] The m-Endo method is also used to determine the quantity of E.coli and general coliforms and is an official USEPA approved method fortesting water quality. The medium is commonly used with a membranefilter and E. coli and general coliform colony forming units (CFU) growas dark colonies with a golden green metallic sheen. Due to a provenhigh rate of false positive error, typical colonies must be confirmed byadditional testing. Standard Methods for the Examination of water andWastewater, 20^(th) Edition, 9-10 &9-60 (1998).

[0009] Other tests, such as the Most Probable Number (MPN), utilizelactose containing broths (LST, BGLB, EC) to estimate numbers of generalcoliforms and E. coli, but have also been shown to have high rates orerror as well as being cumbersome and slow to produce results. Evans etal., Appl. Envir. Microbiol. 41:130-138 (1981).

[0010] The reagent 5-bromo-4-chloro-3-indolyl-β-galactopyranoside(X-gal) is a known test compound for identifying coliforms. When actedon by the β-galactosidase enzyme produced by coliforms, X-gal forms aninsoluble indigo blue precipitate. X-gal can be incorporated into anutrient medium such as an agar plate, and if a sample containingcoliforms is present, the coliforms will grow as indigo blue colonies.X-gal has the advantage over the compound ONPG, described above, in thatit forms a water insoluble precipitate rather than a diffusiblecompound, thereby enabling a quantitative determination of coliforms tobe made when the test sample is incorporated into or onto a solidifiedmedium, or when coliform colonies grow on the surface of a membranefilter resting on a pad saturated with a liquid medium or on a membranefilter resting on a solid medium. Further, it does not require the useof ultraviolet light.

[0011] A similar compound, 5-bromo-4-chloro-3-indolyl-β-D-glucuronide(X-gluc) is a known test compound for identifying E. coli. When acted onby the β-glucuronidase enzyme produced by most E. coli, X-gluc forms aninsoluble indigo blue precipitate. X-gluc has the advantage over thecompound MUG, described above, in that it forms a water insolubleprecipitate, rather than a diffusible compound, thereby enabling aquantitative determination of E. coli to be made when the test sample isincorporated into or onto a solidified medium. X-gluc and its ability toidentify E. coli are described in Watkins, et al., Appl. Envir.Microbiol. 54:1874-1875 (1988). A similar compound,indoxyl-β-D-glucuronide, which also produces sharp blue colonies of E.coli, was described in Leg, et al., Can. J. Microbiol. 34:690-693(1987).

[0012] Although X-gal and X-gluc are each separately useful in thequantitative determination of either coliforms (X-gal) or E. coli(X-gluc), these indicator compounds have the disadvantage that they eachcontain the same chromogenic component. Therefore, they cannot be usedtogether to identify and distinguish both E. coli and general coliformsin a single test with a single sample, since they both generateidentically hued indigo blue colonies. A person using both reagentstogether would be able to quantitatively identify the total number ofcoliforms, the same as if X-gal were used alone, but would not be ableto indicate which of the colonies were E. coli and which were othercoliforms besides E. coli.

[0013] A recently developed and highly commercially successful testmethod and test medium for quantitatively identifying anddifferentiating general coliforms and E. coli in a test sample isdescribed in U.S. Pat. Nos. 5,210,022, and 5,393,662, both of whichshare common inventorship with the present application and which arehereby incorporated by reference. This method and test medium improvesupon prior art methods by allowing the quantitative identification ofgeneral coliforms and E. coli in a single sample. Additionalconfirmatory tests are not necessary. The test sample is added to amedium containing a β-galactosidase substrate, such as6-chloroindolyl-β-D-galactoside, and a β-glucuronidase substrate, suchas 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc). Theβ-galactosidase substrate is capable of forming a water insolubleprecipitate of a first color upon reacting with β-galactosidase, and theβ-glucuronidase substrate is capable of forming a water insolubleprecipitate of a second color, contrasting with the first color, uponreacting with β-glucuronidase. As a result, general coliforms may bequantified by enumerating the colonies of the first color (havingβ-galactosidase activity), and E. coli may be quantified by enumeratingthe colonies of the second color (having both β-galactosidase andβ-glucuronidase activity). This technology has been widely copied.

[0014] Another recently developed test method and apparatus providesexcellent results for the differentiation and identification of generalcoliforms, E. coli and E. coli 0157 strains and non-coliformEnterobacteriaceae. The method and test medium are described in U.S.Pat. No. 5,726,031, which shares common inventorship with the presentapplication, and which is hereby incorporated by reference.

[0015] A certain class of substrates, referred to herein as“nonchromogenic,” have been used to detect various microorganisms. Adipslide for detecting E. coli using hydroxy-quinoline-β-D-glucuronideis disclosed by Dalet et al., J. Clin. Microbiol, 33(5):1395-8 (1995).Similarly, a technique for detection of E. coli in an agar-based mediumusing 8-hydroxyquinoline-β-D-glucuronide is disclosed by James et al.,Zentralbl Bakteriol Mikrobiol Hyg [A], 267(3):316-21 (1988).

[0016] It is desirable to further improve the distinguishing colorsgenerated in the test media. That is to say, in prior art test mediawhich detect and distinguishing two biological entities, confusion mayarise between the two colors which show in the media.

[0017] Further, it is desirable to be able to identify and differentiateother closely related organisms, such as members of the familiesAeromonaceae, Vibrionaceae, and Salmonella. For example, the genusAeromonas is closely related to coliforms and gives an almost identicalbiochemical test pattern. Further, the genus Vibrio is also an importanttype of bacteria that grows under the same general conditions ascoliforms. It is known to distinguish Aeromonas colonies from generalcoliforms by testing all colonies in a given sample for the presence ofcytochrome oxidase. Undesirably, however, this requires another set oftests. Further, Aeromonas is common in water and food, and it iscommonly indicated in test samples as general coliforms, which resultsin high a false positive error for general coliforms by most currenttest methods. The Aeromonas can be prevented from interfering with thecoliform results by adding certain antibiotics to the medium. However,the antibiotic amounts added must be carefully controlled. Further, theantibiotics which have been conventionally used have short life spans inthe media so that they lose their potency quickly in other than a frozencondition. It may often be desirable to be able to culture, identify andenumerate Aeromonas spp. which cannot be done if they are inhibited.

[0018] Further, in those cases where it is desirable to inhibitAeromonas, it is desirable for a better method of so doing, one in whichthe shelf life of the medium is not appreciably reduced by the inclusionof an inhibitor.

[0019] Additionally, it is also desirable to distinguish strains ofSalmonella from E. coli, general coliforms and Aeromonas.

SUMMARY OF THE INVENTION

[0020] The present invention overcomes the disadvantages of prior artmethods by providing a test method and medium for quantitatively orqualitatively identifying and differentiating biological entities in atest sample that may include a plurality of different biologicalentities.

[0021] In one embodiment, the present invention introduces the use of“nonchromogenic” substrates to enhance the distinction among multiplecolors produced by distinct biological entities present in the inventivetest medium. Unexpectedly, it has been discovered that other“chromogenic” substrates present in the inventive test medium do notinterfere with the substantially black color achieved with thenonchromogenic substrate. That is to say, so long as a given biologicalentity is responsive to the nonchromogenic substrate, aggregationsthereof present in the test medium will show as a substantially blackcolor—independent of whether such biological entity is responsive toone, two or more chromogenic substrates which are also present in themedium. The present invention exploits this hitherto unexplored propertyof nonchromogenic substrates.

[0022] In one form thereof, the present invention provides a test mediumfor detecting, identifying and qualifying or quantifying first andsecond biological entities. The test medium includes a nutrient basemedium including ions of a salt, a chromogenic substrate and anonchromogenic substrate. The first biological entity is responsive tothe nonchromogenic substrate whereas the second biological entity isresponsive to the chromogenic substrate. In this test medium,aggregations of the first biological entity present in the test mediumare substantially black and aggregations of the second biological entitypresent in the test medium are a second color, the second color beingdistinguishable from the substantially black.

[0023] In one form, the inventive test medium accounts for the firstbiological entity being responsive to the chromogenic substrate inaddition to the nonchromogenic substrate. In such event, aggregations ofthe first biological entity present in the test medium will nonethelessshow as substantially black.

[0024] Significantly, even though the aggregations of the firstbiological entity are responsive to both the first and second substratesin this form, these aggregations still show as substantially black inthe test medium. That is, the chromogenic substrate does not interferewith the substantially black color. Advantageously, this property ofnonchromogenic substrates allows several different biological entitiesto be identified and differentiated in a single medium, aggregations ofeach biological entity having a visually distinguishable color.

[0025] In another form of the above-described inventive medium, themedium further includes the antibiotic nalidixic acid to inhibit thegrowth of Aeromonas, spp. Advantageously, it has been found thatnalidixic acid, as compared with cefsulodin, does not significantlyreduce the shelf life of the test medium incorporating it.

[0026] In this connection, another form of the present inventionprovides a method of making a test medium for detecting at least onefirst type of biological entity and inhibiting a second type ofbiological entity from growing in the medium. The method includes thesteps of combining desired substrates with a nutrient base medium;adding an inhibitor to the medium; and then sterilizing the medium bysubjecting the medium to at least 100° C. Because the inhibitor is addedas an initial step, subsequent sterile addition of inhibitor isunnecessary.

[0027] In another form thereof, the present invention provides a testmedium for detecting, identifying and qualifying or quantifying first,second and third biological entities. The test medium includes anutrient base medium including ions of a salt. First and secondchromogenic substrates and a nonchromogenic substrate are provided inthe test medium. The first and second biological entities are responsiveto the first and the second chromogenic substrates, respectively, andthe third biological entity is responsive to the nonchromogenicsubstrate. Aggregations of the first biological entity present in thetest medium are a first color, aggregations of the second biologicalentity present in the test medium are a second color, and aggregationsof the third biological entity present in the test medium aresubstantially black.

[0028] In one form, the inventive test medium accounts for the thirdbiological entity being responsive to the first and/or the secondchromogenic substrates in addition to the nonchromogenic substrate. Insuch event, aggregations of the third biological entity will nonethelessshow as substantially black.

[0029] It should be appreciated that the use of a nonchromogenicsubstrate along with one or more chromogenic substrates synergisticallyincreases the number of biological entities that can be detected anddistinguished in a single medium and synergistically increases thepossible color combinations for a given set of biological entities to bedetected. Stated another way, including a nonchromogenic component asone of the substrates synergistically increases the degrees of freedomin selecting other substrates and corresponding colors for a testmedium. This is so because an aggregation of the biological entity whichis responsive to the nonchromogenic substrate will dependably show assubstantially black. No combined color effects need be accounted forwith the nonchromogenic substrates. For example, in a test mediumincluding three chromogenic substrates and a nonchromogenic substrate,at least three combined color combination effects are avoided by usingthe one nonchromogenic component, as compared with using fourchromogenic components.

[0030] The present invention, in another form thereof, provides a testmedium capable of detecting, quantifying, and differentiating generalcoliforms and/or E. coli spp. under ambient light. The test mediumcomprises a nutrient based medium including a salt. A first substratecapable of forming a first water insoluble component of a first color inthe presence of E. coli and the ions of the salt is provided in themedium. The first color is substantially black. A second substratecapable of forming a second water insoluble component of a second colorin the presence of general coliforms is provided. The second color isvisually distinguishable from the first color. Thus, colonies of E. colipresent in the test medium are indicated by the first substantiallyblack color and colonies of general coliforms are indicated by thesecond color.

[0031] In one form of the above invention, the test medium furtherincludes a third substrate capable of forming a third water insolublecomponent of a third color in the presence of Salmonella. The thirdcolor is distinguishable from the first and second colors, whereby thetest medium is capable of quantifying and/or differentiating E. coli,general coliforms and Salmonella. Further, the substrates are selectedsuch that general coliforms present in the test medium will also reactwith the third substrate to form a water insoluble component whichincludes the third color. Consequently, general coliform colonies areindicated in the test medium as a fourth color, the fourth color being acombination of the second color and the third color. The fourth color isvisually distinguishable from the first and third colors. Still further,the substrates can be selected such that Aeromonas spp. form aninsoluble component of the second color by reacting with the secondsubstrate, but not the first and third substrates. Thus, in theinventive test medium, E. coli colonies will be generally black, generalcoliform colonies will be the fourth color, Aeromonas colonies will bethe second color and Salmonella colonies will be the third color.

[0032] In another form thereof, the present invention provides a methodfor detecting, quantifying and differentiating under ambient lightgeneral coliforms, E. coli, and at least one of the genera Aeromonas orSalmonella in a test sample. The method comprises the steps of providinga nutrient base medium including first, second and third substrates.Each of the substrates is capable of forming a water insoluble componentin the presence of at least one of general coliforms, E. coli,Aeromonas, Salmonella. The substrates are selected such that colonies ofE. coli produced in the test medium are a first color, colonies ofgeneral coliforms produced in the test medium are a second color, andcolonies of one of Aeromonas and Salmonella produced in the test mediumare a third color. Each of the colors are visually distinguishable. Thetest medium is inoculated with the test sample and then incubated. Thetest medium is then examined for the presence of first colonies havingthe first color, second colonies having the second color, and thirdcolonies having the third color. The first colonies are E. coli, thesecond colonies are general coliforms, and the third colonies are one ofAeromonas or Salmonella.

[0033] In one form thereof, the inventive method further includes addingions from a salt to the test medium to react with one or more of thesubstrates. In so doing, a precipitate is produced which shows as asubstantially black color in the presence of the specific enzyme forthat substrate. A preferred compound for forming the substantially blackcolor in the presence of the ions of the salt consists of aβ-D-glucuronide. These compounds release an aglycone when hydrolizedwhich forms a substantially black insoluble complex in the presence ofions.

[0034] In another form of the inventive method, the method furthercomprises examining the test medium for the presence of fourth colonieshaving a fourth color, wherein the substrates are selected such thatcolonies of Aeromonas are the third color and colonies of Salmonella arethe fourth color, the fourth color being visually distinguishable fromthe first, the second and the third colors. The substrates may beselected such that the first color is substantially black, the secondcolor is substantially blue-violet, the third color is substantiallyred-pink and the fourth color is substantially teal-green.

[0035] In another form of the inventive method, the substrates areselected such that colonies of Aeromonas as well as colonies ofPlesiomonas and Vibrios are indicated as the third color.

[0036] One embodiment of the present invention uses a nonchromogenicsubstrate along with one or more chromogenic substrates and therebysynergistically increases the degrees of design freedom in selectingcolors for the inventive test medium. This is so because the chromogenicsubstrates do not interfere with the substantially black precipitateformed by the nonchromogenic substrate.

[0037] Another advantage of one embodiment of the present invention isthat it enables the quantification, identification and differentiationof four (4) different bacterial strains simultaneously in a single testmedium using a single test sample, under ambient lighting. Subsequenttests with their concomitant extra time spent and extra costs areavoided. Of course, the inventive test medium of the present inventioncould also be used purely for qualitative purposes, as a merepresence/absence (P/A) test.

[0038] In one embodiment, the substrates are selected such that thecolors are easy to visually distinguish from one another without theneed for UV light or other visual aids, other than, perhaps,magnification means. For example, in one embodiment, E. coli coloniesare clearly indicated by a precipitate having a substantially blackcolor, general coliform colonies are indicated by a blue-violet color,Aeromonas colonies are indicated by a red-pink color, and Salmonellacolonies are indicated by a teal-green color. Because these colors arevisually so distinct, confusion among the colors is greatly reduced ascompared to prior art media. In one embodiment, MUGluc(4-methylumbelliferyl-Beta-D-glucuronic acid) is used in place of anonchromogenic substrate. In this test medium, general coliforms wouldstill be indicated by a blue-violet or grayish color. Aeromonas coloniesindicated by a red-pink color and Salmonella colonies indicated by ateal-green color; however, E. coli colonies would look the same invisible light as general coliforms, but also would fluoresce a brightbluish color under a long wave UV light and this could be distinguishedfrom the other colonies. Although, the fluorescent product would diffusemore quickly than chromogenic or nonchromogenic substrates and makequantifying the colonies of E. coli more difficult, the E. coli coloniescan be detected at around 14 hours incubation time, and in any case willsuffice as a presence/absence test for the E. coli.

[0039] In yet another embodiment of the invention, it has been foundthat three chromogenic substrates may be used if properly combined. Forexample, a β-glucuronide such as X-Gluc(5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid) or Iodo-Gluc(5-iodo-3-indolyl-β-D-glucuronic acid) may be used with chromogenic α-and β-D-galactoside substrates. Examples of β- and β-galactosidesubstrates that may be suitable are 6-chloro-3-indolyl-β-D-galactosideand 5-bromo-4-chloro-3-indolyl-α-D-galactoside. In this embodiment, theβ-D-glucuronide and α-D-galactoside substrates form the same generalcolor in the presence of colonies that produce the respective enzymes;however, the colors may be distinguished by providing the substrates indifferent amounts so that the resulting color produced by one is darkerthan that produced by the other. In addition, even if the substrates areprovided in approximately the same amounts, colonies that react to boththe β-D-glucuronide and α-D-galactoside, such as E. coli should bedarker that colonies such as general coliforms, which only react to theα-D-galactoside It should be appreciated, that it would not be necessaryto add ions of salt if a nonchromogenic substrate is not used.

[0040] In an additional aspect of the present invention, MUGluc oranother fluorescent glucuronide substrate may be combined in a testmedium with a chromogenic or nonchromogenic glucuronide substrate. Inthis case, the MUGluc substrate can be used to detect E. coli underfluorescent light with less incubation time than is required to detectcolonies with a chromogenic or nonchromogenic glucuronide. In addition,the MUGluc substrate can serve as a confirmation of the presence/absenceof E. coli, if for any reasons there is some question as to the colorsvisible in ambient light produced by the colonies in the presence of thesubstrates.

[0041] Another advantage of the test medium of the present invention isits flexibility and ease of use. The incubation temperature is notcritical as growth and differentiation of the organisms mentioned mayoccur within an optimum range. Therefore, resuscitation steps areavoided and inhibition of temperature sensitive strains does not occur.Also, inexpensive equipment may be used.

[0042] In one embodiment of the present invention, the color distinctionobtained in a test medium can be intensified for identifying anddifferentiating E. coli from general coliforms. In one test medium, E.coli colonies present a substantially black color, whereas generalcoliforms present a red-pink color, the distinction therebetween beingmuch more apparent than in prior art test media. Confusion between thetwo colors is therefore greatly reduced.

[0043] Still another advantage of the present invention is that itenables the identification and differentiation of Aeromonas spp. fromgeneral coliforms. Prior art test media undesirably require using acefsulodin inhibitor for preventing Aeromonas spp. from growing therein.However, the use of cefsulodin as an inhibitor requires an extra step inthe process, viz., sterile addition of filter sterilized antibiotic, andis difficult to control. Further, the presence of cefsulodinsignificantly reduces the effective shelf life of the medium. Further,the use of an inhibitor, obviously, prevents the detection andquantitification of Aeromonas spp. Advantageously, with the presentinvention, Aeromonas spp. can be detected, quantified and differentiatedfrom general coliforms in a single medium.

[0044] As a related advantage, if it is nonetheless desired to inhibitcolonies of Aeromonas spp. from growing in the test medium, the presentinvention provides a superior means for doing so. Specifically,preferred forms of the present invention employ nalidixic acid as aninhibitor, which has been shown to have a far less deleterious effect tothe shelf-life of the medium incorporating it. Further, nalidixic acidcan be added as part of the initial medium formulation prior tosterilization, thereby avoiding a costly and difficult process stepwhich is required with cefsulodin. Finally, nalidixic acid is much lessexpensive than cefsulodin.

[0045] Another advantage of the present invention is that it can providea test medium for qualitative or quantitative testing. That is, the testmedia in accordance with the present invention can be used as merepresence/absence test devices, or can be used to quantify variousbiological entities showing as different colored colonies on theinventive test media.

DETAILED DESCRIPTION OF THE INVENTION

[0046] The method and medium of the present invention allow thesimultaneous detection, quantification, identification anddifferentiation of a variety of selected biological entities in a sampleof mixed populations of biological entities. The inventive method andmedium are particularly useful for the detection, quantification,identification and differentiation of E. coli and general coliforms, andfurther quantitative identification and differentiation of otherselected biological entities, including Aeromonas, Salmonella, andVibrio bacterial species.

[0047] The method and test media incorporating the present inventionutilize the fact that the enzymatic activity of biological entities andspecifically of bacteria varies with the genus, and/or family ofbacteria of interest. The method and test media incorporating thepresent invention further utilize the fact that various enzymeidentifying substrate complexes can be used to identify specific enzymeswith the production of distinctive colors. Significantly, in oneembodiment of the present invention, the method and test mediaincorporating the present invention exploit the fact that chromogenicsubstrates present in a test medium do not interfere with thesubstantially black color produced by nonchromogenic substrates.

[0048] While nonchromogenic substrates are known in the art, per se,their distinct properties vis-à-vis chromogenic substrates have beenunrecognized. However, the behavior of a nonchromogenic substrate in amedium including the combination of chromogenic substrates is unique. Toillustrate, aggregations of a biological entity which are responsive totwo chromogenic substrates will typically show in a test medium as acombination of the two colors produced upon cleavage of the tworespective substrates. When three chromogenic substrates are involved,as in another embodiment of the invention, the combined color effect isnot obvious to predict and account for. Further, inherent variations inthe amount of enzymes produced by particular strains of biologicalentities can result in different shades or hues of colors upon cleavageof the chromogenic substrates. Consequently, the colors can be difficultto distinguish for the lay person examining the test medium. Chromogenicsubstrates must therefore be chosen and used in a concentration in viewof the other chromogenic substrates planned for inclusion in a giventest medium.

[0049] Such is not the case with the nonchromogenic components. Whileaggregations of biological entities which are responsive to chromogenicsubstrates in addition to nonchromogenic substrates may show in the testmedium as having a colored or fluoroescent “halo,” such aggregationsnonetheless appear substantially black and are therefore easy toidentify. Multiple “degrees of freedom” are achieved with thenonchromogenic components.

[0050] Using a nonchromogenic substrate is one way of enabling a singletest medium to differentiate four (4) different bacterial strains withfour (4) visually distinguishable colors. The black color is difficultto mistake. Further, the substantially black pigmentation does notdiffuse so that the location of the colonies is precisely known and thecolonies can be accurately counted. The nonchromogenic substratesproduce an insoluble chelated compound which is different than the dimerwhich is produced by the chromogenic substrates.

[0051] The inventive test medium and method allows not only a detection,quantification or qualitative identification and differentiation ofgeneral coliforms and E. coli, but also of Salmonella and Aeromonas, aswell as Plesiomonas and Vibrio. Plesiomonas and Vibrios species aredetermined but not differentiated from Aeromonas species as they arevery closely related.

[0052] Definitions

[0053] Biological entities, such as general coliforms, E. coli., etc.,are herein referred to as being “responsive” to certain chromogenic andnonchromogenic substrates. More specifically, a biological entity willpredictably produce specific enzymes when the entity is present in atest medium such as the one described hereinbelow. These enzymes willselectively cleave chromogenic and nonchromogenic substrates. Uponcleavage, these substrates produce a color in the test medium. Themechanism for producing the color is different for chromogenic andnonchromogenic substrates, as described hereinbelow.

[0054] Microorganisms having β-galactosidase activity include thosecommonly known by the designation “coliform.” There are variousdefinitions of “coliform,” but the generally accepted ones includebacteria which are members of the Enterobacteriaceae family, and havethe ability to ferment the sugar lactose with the evolution of gas andacid. Most coliforms are positive for both α-and β-galactosidase. Thatis, they produce both α-and β-galactosidases.

[0055] Microorganisms having β-glucuronidase activity in addition togalactosidase activity primarily include most strains of Escherichiacoli. That is, E. coli is positive for both α- and β-galactosidase aswell as β-glucuronidase.

[0056] The term “general coliforms” as used in this application refersto coliforms other than the various strains of E. coli. These “generalcoliforms” are gram-negative, non-spore forming microorganisms generallyhaving α- and β-galactosidase activity (i.e., lactose fermenters), butnot having β-glucuronidase activity, and having the ability to fermentthe sugar sorbitol.

[0057] For purposes of this specification, a “chromogenic substrate” isa substrate which needs no additional chemicals present in the testmedium upon hydrolysis for color production. That is, a chromogenicsubstrate is cleaved by the specific enzyme corresponding to thatsubstrate to form a dimer with the color being concentrated in the areaof cleavage of the substrate. Many chromogenic substrates are known inthe art. For purposes of this specification “chromogenic” includesfluorogenic substrates. The products of fluorogenic substrates requireultraviolet (UV) light to be detected and are more water soluble thanother chromogenic substrates.

[0058] Certain substrates, referred to herein as “nonchromogenic,”produce a dark, substantially black precipitate in the presence of ionsof a salt and enzyme activity. For example,8-hydroxyquinoline-β-D-glucuronide, when included in a medium along witha salt that produces ions, such as ferric ammonium citrate, will producea substantially black precipitate in the presence of β-glucuronidaseproduced by E. coli or other biological entities. More specifically,upon cleavage of the nonchromogenic substrate by the particular enzyme,a substantially black water-insoluble complex forms in the medium. Thesubstantially black precipitate consists of the ferric ions and theaglycone released when the substrate is hydrolized by the glucuronidasefrom E. coli. This precipitate is a chelated compound which does notdiffuse. Nor is the substantially black color susceptible tointerference from chromogenic compounds present in the test medium.

[0059] For purposes of this specification a “nonchromogenic substrate”means that a chemical in addition to those used with chromogeniccomponents must be present in the test medium when the substrate iscleaved by its corresponding enzyme. The substantially black precipitateformed thereby is a combination of the substrate—salt complex and is nota dimer as is formed by the “chromogenic compounds.”

[0060] For purposes of this specification, the expression “under ambientlight” refers to the visible spectrum, i.e., colors which can be seenand distinguished with the naked eye. A colony present in a test mediumwhich requires ultraviolet light to be seen, for example, would not fallunder the definition “under ambient light”. However, it is to beunderstood that the term “under ambient light” includes using amagnification device, if necessary. Magnification can be especiallyhelpful when counting numerous colonies. The term “visuallydistinguishable” refers to two or more colors which can bedifferentiated under ambient light.

[0061] For purposes of this specification, the term “substantiallyblack” includes dark brown to black, and also includes black withvarious colored halos, such as red-violet, green, fluorescent, etc.

[0062] For further purposes of this specification, color names recitedherein are given as guidance, but it is to be understood that the colornames are to be read broadly. That is, there can be overlap among therecited colors. This is because, as discussed, biological entitiesproduce varying amounts of enzymes, which in turn affects the shade orhue of the resulting color.

[0063] The term “β-galactosidase substrate” as used herein refers to aβ-galactoside comprising galactose joined by β-linkage to a substituentthat forms a detectable compound when liberated by the action ofβ-galactosidase on the substrate. Similarly, the term “α-galactosidasesubstrate” as used herein refers to α-galactoside comprising galactosejoined by α-linkage to a substituent that forms a detectable compoundwhen liberated by the action of α-galactosidase on the substrate. Theterm “β-glucuronidase substrate” as used herein refers to aβ-glucuronide comprising glucuronic acid joined by β-linkage to asubstituent that forms a detectable precipitate when liberated by theaction of β-glucuronidase on the substrate.

[0064] The α- and β-galactosidase substrates and compounds and any othersubstrates described herein as well as the β-glucuronidase substratesand compounds and any other substrates described herein may comprisecarboxylate salts formed by reacting a suitable base with theappropriate galactoside or glucuronic carboxyl group. Suitable basesinclude alkali metal or alkaline earth metal hydroxides or carbonates,for example, sodium hydroxide, potassium hydroxide, calcium hydroxide,magnesium hydroxide, and corresponding carbonates; and nitrogen basessuch as ammonia, and alkylamines such as trimethylamine, triethylamineand cyclohexylamine.

[0065] Designing a Test Medium for Specific Biological Entities

[0066] Certain members of the family Enterobacteriaceae can bedistinguished by the presence of α-galactosidase activity in the absenceof β-galactosidase activity, or vice-versa. For example, most Salmonellaand Shigella spp. are positive for α-galactosidase, but negative forβ-galactosidase. Similarly, Aeromonas spp. can be distinguished fromother members of the family Enterobacteriaceae by the presence ofβ-galactosidase activity in the absence of α-galactosidase activity. Themethod and medium incorporating the present invention are designed totake advantage of these distinguishing characteristics. For example, thespecificity of enzyme activity for Salmonella and Aeromonas spp., asopposed to general coliforms, can be exploited, as illustrated below.

[0067] The method described herein is particularly suitable for thedetection, quantification or qualitative identification anddifferentiation of the different classes of microorganisms describedpreviously, viz., general coliforms, E. coli, Aeromonas and Salmonella.Although the inventive method is particularly suitable for theseparticular microorganisms, it is not limited thereto. Instead, thetechniques described herein have application to the identification anddifferentiation of a wide variety of biological entities.

[0068] That is, specific biological entities are “responsive” to varioussubstrates. More particularly, these biological entities predictablyproduce or contain known enzymes. Substrates, either chromogenic ornonchromogenic, can be selected which, in the presence of a particularenzyme(s), will form a product of a predictable and distinguishablecolor. Multiple substrates can be selected to simultaneously identify aplurality of distinct biological entities in a single test medium,aggregations of each distinct entity being identifiable by a separate,distinguishable color. Further, while certain embodiments disclosedherein distinguish all of the various aggregations present in a testmedium under ambient light, as that term is defined herein, such is notnecessary. For example, several substrates disclosed herein require theuse of ultraviolet light for the aggregations present in the medium tobe seen.

[0069] Table I lists various enzymes whose presence may be detectedusing certain of the substrates listed in Table II. TABLE I Enzymes andAbbreviations Aara = α-D-arabinopyranosidase Bglu =β-D-glucopyranosidase Agal = α-D-galactopyranosidase Bgluc =β-D-glucuronidase Aglu = α-D-glucopyranosidase Bman =β-D-mannopyranosidase Bcel = β-D-cellopyranosidase Bxyl =β-D-xylopyranosidase Bfuc = β-D-fucopyranosidase Nagal = N-acetyl-β-Bgal = β-D-galactopyranosidase D-galactopyranosidase Afuc =α-D-fucopyranosidase Naglu = N-acetyl-β- Bxyl = β-D-xylopyranosidaseD-glucopyranosidase Aman = α-D-mannopyranosidase Bara =β-D-arabinopyranosidase Axyl = α-D-xylopyranosidase Acel =α-D-cellopyranosidase esterase Agluc = α-D-glucuronidase Nagluc =N-acetyl-β-D-glucuronidase

[0070] TABLE II Various Substrates and Color Upon Cleavage6-chloro-3-indolyl substrates Pink 5-bromo-4-chloro-3-indolyl substratesTeal 3-indolyl substrates Teal N-methylindolyl substrates Greennitrophenyl substrates Yellow nitroaniline substrates Yellow8-hydroxyquinoline substrates (and ion of salt) Substantially blackcyclohexenoesculetin substrates (and ion of salt) Substantially blackesculetin substrates (and ion of salt) Substantially black quinolinesubstrates (and ion of salt) Substantially black 5-Iodo-3-Indolylsubstrates Purple 5-Bromo-6-Chloro-3-Indolyl substrates Magenta6-Fluoro-3-Indolyl substrates Pink coumarin substrates Fluorescentfluorescein substrates Fluorescent rhodamine substrates Fluorescentresorufin substrates Fluorescent

[0071] Specific substrate compounds applicable for use with the testmedium of the present invention are available as follows:

[0072] 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) is acommercially available β-galactosidase substrate that produces aninsoluble precipitate having an approximately teal color when reactedupon by β-galactosidase and is available from Biosynth International,Naperville, Ill.

[0073] 6-chloro-3-indolyl-β-D-glucuronide is a compound which producesan insoluble precipitate having a magenta color, the preparation ofwhich is described in the aforementioned incorporated by reference U.S.Pat. No. 5,210,022 and is available from Research Organics, Cleveland,Ohio.

[0074] The compound 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc)is a commercially available β-glucuronide that produces an insolubleprecipitate having an approximately teal color when reacted upon byβ-glucuronidase. Similarly, indoxyl-β-glucuronide is a similar compound,the preparation of which is described in the aforementioned article byLeg et al., in Can J. Microbiol., the disclosure of which isincorporated by reference.

[0075] Another suitable β-galactoside is the compound6-chloro-3-indolyl-β-D-galactoside which produces an insolubleprecipitate having a pink/magenta color, the preparation of which isdescribed in the aforementioned U.S. Pat. No. 5,210,022.

[0076] Other suitable compounds applicable as substrates in the practiceof the present invention are specified in U.S. Pat. No. 5,210,022, allof which are incorporated herein by reference.

[0077] The substrate 8-hydroxyquinoline-β-D-glucuronide is acommercially available β-glucuronide that, in the presence of metallicions such as iron, produces an insoluble precipitate having asubstantially black color when reacted upon by β-glucuronidase and inthe presence of other α- or β- galactoside substrates.8-hydroxyquinoline-β-D-glucuronide is available from BiosynthInternational, Naperville, Ill.

[0078] Further, a salt providing ions suitable for use with the presentinvention is ferric ammonium citrate, available from Sigma Chemical, St.Louis, Mo. The cyclohexenoesculetin substrates are described in James etal., Appl. & Envir. Micro. 62:3868-3870 (1996) and in the presence offerric ions, produce an insoluble substantially black precipitate.

[0079] N-methyl-indolyl substrates such asN-methylhydroxy-β-D-galactopyranoside are commercially available fromBiosynth International, Naperville, Ill.

[0080] Nitrophenyl substrates, such as2-nitrophenyl-β-D-galactopyranoside, are commercially available fromBiosynth International, Naperville, Ill. Similarly, nitroanilinecompounds are available for synthesis through Sigma Chemical, St. Louis,Mo.

[0081] Other substrates producing a substantially black color includeesculetin substrates such as cyclohexenoesculetin-β-D-galactoside, whichis described in James et al., Appl. & Envir. Microbiol. 62:3868-3870(1996). Quinoline substrates, such as8-hydroxyquinoline-β-D-galactopyranoside and8-hydroxyquinoline-β-D-glucuronide are available through BiosynthInternational, Naperville, Ill.

[0082] Iodo-indolyl substrates, such as5-iodo-3-indolyl-β-D-galactopyranoside are available through BiosynthInternational, Naperville, Ill.

[0083] Several fluorescent substrates are suitable for use with thepresent invention. Coumarin substrates such as 4-methylumbelliferylsubstrates and 5-trifluoromethylumbelliferyl substrates are commerciallyavailable from Biosynth International, Naperville, Ill. Also suitableare fluorescein substrates, rhodamine substrates, and resorufinsubstrates. No commercial source is known for these three substrates butcomponents are available from Sigma Chemical, St. Louis, Mo.

[0084] While specific examples of substrates suitable for use with thepresent invention have been enumerated hereinabove, such is not to beconstrued as limiting the invention in any manner. Instead, one ofordinary skill in the art can use Table IV and V hereinbelow to identifya virtually limitless number of substrates.

[0085] Preparation of Test Medium

[0086] The test medium is formed by combining the desired substrateswith a nutrient base medium. The nutrient base medium can be any culturemedium known in the art for providing the maintenance and reproductionof living cells. Generally, such media include nutrients, buffers,water, and sometimes a gelling agent. Possible gelling agents includeagars, pectins, carrageenans, alginates, locust bean, and xanthins,among others.

[0087] The following is an example of the preparation of a test mediumsuitable for use in this invention. This example coincides with ExampleI, below.

[0088] The substrates 8-hydroxyquinoline-β-D-glucuronide,5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside, and6-Chloro-3-indolyl-β-D-galactopyranoside are added in quantities of 250mg/L medium; 70 mg/L medium; and 175 mg/L medium, respectively. Thesubstrates are added directly to the hot (75°-85° C.) medium (formulabelow) in a blender prior to sterilization.

[0089] Standard agar medium may be made by adding 15 gm ofbacteriological quality agar gum to the following nutrient formulaPancreatic Digest of Casein  5.0 gm Yeast Extract  3.0 gm DipotassiumPhosphate   .3 gm Deionized Water 990 ml Ferric Ammonium Citrate 800 mgin 10 ml deionized water (sterilized separately from the othercomponents)

[0090] and then sterilizing at 121° C. for 15 minutes. The medium shouldbe adjusted to result in a pH of 7.0. The sterilized agar medium isallowed to drop to a temperature of 45° C. in a water bath and then thesterile solution containing the substrates prepared as described aboveis added. The medium is mixed thoroughly and poured into sterile petriplates at a volume of 20 ml/plate.

[0091] A pectin-based test medium may be prepared using the same stepsdescribed above except that 25 gm of low methoxyl pectin is used as thesolidifying agent and the medium is poured at room temperature intopetri plates containing a thin gel layer containing calcium ions whichcombine with the pectin to form a solid gel. A suitable pectin culturemedium is described in U.S. Pat. No. 4,241,186 and U.S. Pat. No.4,282,317, the disclosures of which are incorporated herein byreference. A pectin-based medium is preferred over a standard agarmedium because it has the advantages of convenience and temperatureindependence for the user. The use of pectin media is well described andaccepted as a result of AOAC collaborative studies and other publishedand in-house investigations.

[0092] A suitable pectin medium is commercially available from MicrologyLaboratories, LLC under the trademark Easygel®. Aqueous based mediumwithout gelling agent is available from Micrology Labs, Goshen Ind., foruse with membrane filters.

[0093] Inoculation of the Test Medium With the Sample

[0094] The test medium may be inoculated with a sample to be tested forthe presence of microorganisms by any method known in the art forinoculating a medium with a sample containing microorganisms. Forexample, the sample to be tested may be added to the petri plates priorto adding the nutrient base medium (pour plate technique) or spread onthe surface of the plates after they have cooled and solidified (swab orstreak plate technique). Liquid samples may also be filtered through amicropore (0.45 micrometer size) membrane filter which is then placed onthe surface of a solid medium or on a pad saturated with the medium.

[0095] Incubation of the Test Medium

[0096] The inoculated test medium is incubated for a sufficient time andat such a temperature for individual microorganisms present in thesample to grow into detectable colonies. Suitable incubation conditionsfor growing microorganisms in a medium are known in the art. Commonly,the test medium is incubated for about 24-48 hours at a temperature ofabout 30°-40° C. Less incubation time may be required, such as about 14hours, to obtain results for a fluorescent substrate.

[0097] Unless inhibitors of the general microbial population are used,the general microbial population as well as general coliforms, E. coli,Aeromonas spp., and Salmonella spp. and Shigella spp. will grow in theincubated test medium. Because the precipitates formed are insoluble(except for the fluorogenic substrates) in the test medium, they remainin the immediate vicinity of microorganisms producing the variousenzymes. As the microorganisms reproduce to form colonies, the coloniesshow as colony forming units having the color produced by the particularsubstrate.

[0098] For example, E. coli produces β-galactosidase andα-galactosidase, but, unlike general coliforms and Aeromonas spp., alsoproduces β-glucuronidase. Therefore, insoluble precipitates of each ofthe β-galactosidase substrate, the α-galactosidase substrate and thenonchromogenic β-glucuronide substrate are formed by the action of therespective enzymes such that colonies of E. coli show as a substantiallyblack color, sometimes having a violet-blue halo therearound.

[0099] General coliforms produce β-galactosidase and α-galactosidase andconsequently cleave both the α-galactosidase and β-galactosidasesubstrates. In the present example, the5-Bromo-4-chloro-3-indolyl-α-D-galactoside substrate produces ablue-green or teal color, whereas the 6-Chloro-3-indolyl-β-D-galactosideproduces a pink, or red-pink color. Thus, general coliform colonies willshow as a blue-violet color, which is a combination of the colorsproduced by each of the α- and β-galactosides, respectively.

[0100] Significantly, however, it has been found that Aeromonas spp.,which are closely related to coliforms, and give an almost identicalbiochemical test pattern, are β-galactosidase positive andα-galactosidase negative. That is, Aeromonas spp. will not hydrolize theα-galactoside substrate. Therefore, Aeromonas colonies present in thetest medium will show as colonies having the pink-red color produced bythe β-galactoside substrate.

[0101] Further, it has been found that members of the genus Salmonellaare positive for α-galactosidase, but negative for β-galactosidase. Thatis, Salmonella will not hydrolize the β-galactosidase substrate.Therefore, colonies of Salmonella present in the test medium will appearas a teal, or blue-green color produced by the α-galactoside substrate.

[0102] Examination of the Test Medium and Enumeration of Microorganisms

[0103] The substrates selected for the above example produce threedistinct colors, and general coliforms are indicated by a fourth colorwhich is a combination of two of the three colors. That is, E. colicolonies show as substantially black, general coliform colonies show asblue-violet, Aeromonas colonies show as red-pink, and Salmonellacolonies show as teal-green. While the individual shades of these colorsmay vary somewhat in the test medium due to factors such as varyingenzyme production of the biological entities, it has been found thatthese four colors are distinct enough so that confusion amongst them isunlikely.

[0104] The colonies of each type of microorganism may be enumerated bycounting the colonies or by other methods known in the art forenumerating microorganisms on a test plate. The number of colonies ofeach type generally indicates the number of microorganisms of each typeoriginally present in the sample before incubation.

[0105] Optional Ingredients

[0106] Inhibitors

[0107] The method of the present invention does not require inhibitors.However, the medium may be made more selective for general coliforms andE. coli if desired by the addition of various compounds that areinhibitory to the general microbial population, but have little or noeffect on coliforms. Following are some compounds which may be used: a)bile salts, about 0.3 g/liter, b) sodium lauryl sulfate, about 0.2g/liter, c) sodium desoxycholate, about 0.2 g/liter, d) Tergitol 7,about 0.1 ml/liter. The use of one or more of these compounds reducesthe background (non-coliform) microorganism presence and makes a lesscluttered plate and eliminates the possibility of inhibition orinterference by the non-coliform organisms in the sample. The use ofcertain antibiotics may accomplish the same result.

[0108] Cefsulodin is commonly used in currently available test media toinhibit Aeromonas spp. However, the use of cefsulodin as an inhibitorrequires an extra step in the process, viz., sterile addition of filtersterilized antibiotic. This step is difficult to control. Further, thepresence of cefsulodin significantly reduces the effective shelf life ofthe medium. It has been found that Nalidixic acid can be used instead ofCefsulodin to inhibit Aeromonas spp. with about the same efficacy.Nalidixic acid is preferable because it can survive the approximately120° C. temperature reached in autoclaving the test media. Therefore,unlike cefsulodin, nalidixic acid can be added to the test media as partof the initial media formulation prior to sterilization (see,preparation of test medium, above). It also follows that the resistanceof the nalidixic acid to unfavorable environmental conditions willresult in a longer shelf life for a medium containing it as compared tocefsulodin.

[0109] Inducers

[0110] It is possible that the enzyme production of the generalcoliforms may be enhanced by the addition to the medium formulations ofvery small amounts of substances known as enzyme inducers. One specificinducer for β-galactosidase is available and is known chemically asisopropyl-β-thiogalactopyranoside. Adding approximately 100 mg/liter ofmedium has a positive and noticeable effect on the speed of enzymeproduction for some species of coliforms. Other enzyme inducers areavailable and may be added to media formulations if enhanced enzymeproduction is deemed helpful.

EXAMPLES

[0111] Listed below are broad examples of test media enzyme substratecombinations to be used in combination with the nutrient formuladiscussed above or other suitable nutrient formulas which may beprepared in practicing the present invention.

[0112] Table III illustrates the flexibility of the preferredembodiments incorporating the present invention. Table III is a matrixof some of the possible four-color combinations available for thepreferred biological entities E. coli, general coliforms, and at leastone of the genera Aeromonas or Salmonella to be detected by using theteachings of this disclosure. Other color combinations are possible. Inmany cases, a plurality of different substrates will achieve a desiredresult, the only difference being the colors detected for a specificenzyme. The preferred color choice for the detection of E. coli isdenoted with an asterisk in Table III, depending on the colors chosen todetect other microorganisms. As discussed above, other chromogenicsubstrates do not interfere with the substantially black color, and thesubstantially black color is easy to distinguish from the other colors.

[0113] As discussed above, the use of Table III requires taking intoaccount the combined color effect discussed above which is produced bythe inclusion of multiple chromogenic substrates in a single medium. Forexample, with reference to the first entry in Table III, it can beunderstood that general coliforms will appear as a combination of (1)red-pink (magenta) and (2) teal, the resulting color being blue-violet.This is the case because general coliforms are responsive to twochromogenic substrates. Similarly, general coliforms will show in a testmedium in accordance with the second entry of Table III as a combinationof (1) red-pink (magenta) and (2) yellow. TABLE III Color possibilitiesfor detection of preferred microorganisms desired color red-pink or darkmagenta teal green yellow black fluorescent fluorescent fluorescentblue/purple light blue/gray 1 general general E. coli E. coli E. coli E.coli coliforms coliforms Aeromonas Salmonella* 2 general E. coli E. coligeneral E. coli E. coli coliforms coliforms Aeromonas Salmonella* 3 E.coli general E. coli general E. coli E. coli coliforms coliformsAeromonas Salmonella* 4 general E. coli general E. coli E. coli E. colicoliforms coliforms Salmonella* Aeromonas 5 E. coli general general E.coli E. coli E. coli coliforms coliforms Salmonella* Aeromonas 6 E. coliE. coli general general E. coli E. coli coliforms coliforms AeromonasSalmonella* 7 E. coli E. coli E. coli E. coli general general E. colicoliforms coliforms Aeromonas Salmonella* 8 general E. coli E. coli E.coli E. coli general E. coli coliforms coliforms Aeromonas Salmonella* 9E. coli general E. coli E. coli E. coli general E. coli coliformscoliforms Aeromonas Salmonella* 10 E. coli E. coli general E. coli E.coli general E. coli coliforms coliforms Aeromonas Salmonella* 11 E.coli E. coli E. coli general E. coli general E. coli coliforms coliformsAeromonas Salmonella* 12 general E. coli E. coli E. coli general E. colicoliforms coliforms Aeromonas Salmonella* 13 E. coli general E. coli E.coli general E. coli coliforms coliforms Aeromonas Salmonella* 14 E.coli E. coli general E. coli general E. coli coliforms coliformsAeromonas Salmonella* 15 E. coli E. coli E. coli general general E. colicoliforms coliforms Aeromonas Salmonella* 16 E. coli E. coli E. coli E.coli E. coli E. coli general general coliforms coliforms AeromonasSalmonella* 17 Aeromonas Salmonella E. coli E. coli general coliforms

[0114] Table IV is a partial list of enzyme patterns for biologicalentities preferred to be to be detected in accordance with the teachingsof this disclosure. It is to be understood that one of ordinary skill inthe art would readily recognized that other enzymes which are known andhave been produced, and enzymes which are known only on a theoreticallylevel, would also perform satisfactorily. TABLE IV GENERAL ENZYME NAMEE. coli COLIFORM Aeromonas Salmonella Plesiomonas Vibrio Aara = α- + +− + D-arabinopyranosidase Agal = α- + + − + D-galactopyranosidase Aglu =α- − + + − + D-glucopyranosidase Bcel = β- − + − − − −D-cellopyranosidase Bfuc = β- + + + − − − D-fucopyranosidase Bgal =β- + + + − + + D-galactopyranosidase Bgal = β- + + + − − −D-glucopyranosidase Bgluc = β- + − − − − − D-glucuronidase Bman = β- + +− + + + D-mannopyranosidase Bxyl = β- − + − − D-xylopyranosidase Nagal =N- − + + − + + acetyl-β-D-galactopyranosidase Naglu =N-acetyl-β-D-glucopyranosidase − + + − + + Aman = α- − − − − − −D-mannopyranosidase esterase = esterase − − − + − −

[0115] Table V is a matrix which teaches a wide variety of substratesand their associated colors for use in test media in accordance with theteachings of this disclosure. The left hand side of Table V indicatesthe color that will result when the listed chromogenic component iscleaved from its corresponding substrate by the specific enzyme for thatsubstrate. In the case of the nonchromogenic components, the color issubstantially black and the reaction mechanism requires the presence ofions of a salt upon cleavage of the substrate, as explained above.

[0116] Test enzymes which are produced by certain biological entities(see Table IV) are found at the right hand side of table V. “Substratecomponents” are shown to the left of the specific test enzymes. Each ofthe substrate components listed on the right hand side of table V can becombined with any of the chromogenic or nonchromogenic components listedon the left hand side of table V to identify a specific substrate foruse in a test medium. It can therefore be understood that Table Vteaches a large quantity of substrates possible for use in accordancewith the present invention. Many of the substrates identified by theabove-described use of table V are commercially available, whereas themethod for producing other identified substrates is described in theliterature. Still other substrates identified by using table V are onlytheoretically possible.

[0117] Nonchromogenic components are included at the bottom left handside of Table V, and are different from the chromogenic componentsbecause they do not form specific colors upon cleavage. Instead, thequinoline or esculetin components combine with ions of a salt (e.g.,ferric salt) which must be present in the medium when the substrate iscleaved by the specific enzyme. The substantially black precipitateformed by the nonchromogenic components is a combination of thequinoline or esculetin—iron complex rather than a dimer which is formedby the chromogenic components.

[0118] Unlike nonchromogenic components, the chromogenic componentsshould be selected in view of all other chromogenic components selectedfor the medium and in view of the enzyme patterns of the entities to bedetected. The selection and concentration of chromogenic componentsshould maximize the distinction among the respective colors produced.

[0119] While many various chromogenic component and substratecomponent/enzyme possibilities are taught by Table V, otherpossibilities within the scope of the appended claims would be possibleby one of ordinary skill in the art. For example, as shown in Table V,one of ordinary skill in the art could combine an N-acetyl group withmany of the sugars of the substrate components listed in Table V. Forexample, an N-acetyl group could be combined with β-D-mannopyranoside toform N-acetyl-β-D-mannosaminide, the corresponding enzyme beingN-acetyl-β-D-mannosaminidase. Any of the chromogenic components ornonchromogenic components listed on the left hand side of Table V couldthen be combined with the substrate component to identify a substrate.If the substrate is commercially available or the method of making it isknown, the substrate could be used in a test medium. Upon cleavage ofthe substrate by the corresponding enzyme in the test medium, the colorlisted will appear.

[0120] Generally, the teachings of this disclosure can be used asfollows to make a test medium for detecting various microorganisms orcell types. First, the microorganisms desired to be detected anddifferentiated are selected. The preferred organisms to be detected areE. coli, general coliforms, and at least one of the genera Aeromonas orSalmonella. Enzymes produced by the selected organisms can be identifiedwith reference to Table IV. Equipped with knowledge of specific enzymesproduced by each microorganism, one can then identify correspondingsubstrates components from the right hand side of Table V. Dependingupon the color desired, one can select a chromogenic or nonchromogeniccomponent from Table V to be combined with the substrate component toidentify a substrate for inclusion in the test medium. If the substratethereby identified is commercially available or the method of itssynthesis is known, the substrate can be used in the test medium. TABLEV COLOR COMPONENT AND SUBSTRATE COMPONENT MATRIX CHROMOGENIC COMPONENT &(COLOR) 6-fluoro-3-indolyl- (pink) 6-chloro-3-indolyl- (pink/red)5-bromo-6-chloro-3-indolyl- (magenta) 3-indolyl- (teal)5-bromo-4-chloro-3-indolyl- (teal) 5-iodo-3-iondolyl- (purple)N-methylindolyl- (green) 4-methylumbelliferyl- (fluorescent) rhodamine-(fluorescent) fluorescein- (fluorescent) resorufin- (fluorescent)coumarin (fluorescent) nitrophenyl- (yellow) nitroaniline (yellow)NONCHROMOGENIC COMPONENT (COLOR) 8-hydroxyquinoline plus ions-(substantially black) 3,4-cyclohexenoesculetin plus ions (substantiallyblack) esculetin plus ions- (substantially black) SUBSTRATE COMPONENTTEST ENZYME α-D-arabinopyranoside Aara. α-D-cellopyranoside Acel.α-D-fucopyranoside Afuc. α-D-galactopyranoside Agal. α-D-glucuronideAgluc. α-D-mannopyranoside Aman. α-D-xylopyranoside Axyl.β-D-arabinopyranoside Bara. β-D-cellopyranoside Bcel. β-D-fucopyranosideBfuc. β-D-galactopyranoside Bgal. β-D-glucopyranoside Bglu.β-D-glucuronide Bgluc. β-D-mannopyranoside Bman. β-D-xylopranoside Bxyl.N-acetyl-β-D-galactosaminide Nagal N-acetyl-β-D-glucosaminide NagluN-acetyl-β-D-glucuronaminide Nagluc N-acetyl + other sugar componentsbutyrate esterase caprylate esterase palmitate esterase

[0121] Table VI is a concise summary of the specific examples. TABLE VIEXAMPLE SUMMARIES General Example # Substrate E. coli ColiformsAeromonas Salmonella* I 8-hydroxyquinoline-β-D-glucuronide X6-chloro-3-indolyl-β-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X color

Black Purple-Blue Pink Teal II 8-hydroxyquinoline-β-D-glucuronide X6-chloro-3-indolyl-β-D-galactopyranoside X X X6-chloro-3-indolyl-β-D-mannoside X X X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X color

Black Purple-blue Red-pink Purple-blue IIIA8-hydroxyquinoline-β-D-glucuronide X6-chloro-3-indolyl-β-D-galactopyranoside X X X6-chloro-3-indolyl-α-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside X X X color

Black Purple-blue Purple-blue Pink IIIB8-hydroxyquinoline-β-D-glucuronide X6-chloro-3-indolyl-β-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside X X X color

Black Purple-blue Purple-blue Teal IIIC May eliminate Aeromonas withinhibitors which allows removal of 6-chloro-3-indolyl-β-D-galactopyranoside from Examples IIIA and IIIB IV6-chloro-3-indolyl-β-D-galactopyranoside X X X6-chloro-3-indolyl-β-D-mannoside X X X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X color

Purple- Purple-blue Pink Purple-blue Blue V-A6-chloro-3-indolyl-β-D-galactopyranoside X X X6-chloro-3-indolyl-α-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside X X X color

Purple- Purple-blue Purple-blue Pink blue V-B6-chloro-3-indolyl-β-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside X X X color

Purple- Purple-blue Purple-blue Teal blue V-C May eliminate Aeromonaswith inhibitors which allows removal of substrate No. 1 from example V-Aand allows removal of substrate No. 3 from example V-B VI5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X6-chloro-3-indolyl-β-D-galactopyranoside X X X color

Purple- Purple-blue Pink Teal blue VII8-hydroxyquinoline-β-D-glucuronide X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X6-chloro-3-indolyl-N-acetyl-β-D-galactosaminide X X Note: In example 7,Vibrio and Plesiomonas also show as pink along with Aeromonas color

Black Purple-blue Pink Teal (see note) VIII8-hydroxyquinoline-β-D-glucuronide X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X6-chloro-3-indolyl-β-D-mannoside X X X6-chloro-3-indolyl-N-acetyl-β-D-galactosaminide X X Note: In example 8,Vibrio and Plesiomonas also show as pink along with Aeromonas color

Black Purple-blue Pink Purple-blue (see note) IX5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X6-chloro-3-indolyl-β-D-galactopyranoside X X X6-chloro-3-indolyl-N-acetyl-β-D-galactopyranoside X X color

Purple- Purple-blue Pink Teal blue X6-chloro-3-indolyl-N-acetyl-β-D-galactopyranoside X X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X6-chloro-3-indolyl-β-D-galactopyranoside X X X Note: For example 10,Vibrio and Plesiomonas also show as pink along with Aeromonas color

Purple- Purple-blue Pink Teal blue (see note) XI8-hydroxyquinoline-β-D-glucuronide X6-chloro-3-indolyl-β-D-galactopyranoside X X X (or)5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside X X X Note: In example11, Aeromonas may be eliminated by adding inhibitors color

Black Pink Pink Not detected Teal Teal or or XII8-hydroxyquinoline-β-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X (or)6-chloro-3-indolyl-α-D-galactopyranoside X X X Note: In example 13,Aeromonas may be eliminated by adding an inhibitor color

Black Black Black Teal or Pink XIII Use same substrates as in exampleNo. 1, and add: Enterobacter and Klebsiella showing as4-methyl-umbelliferyl-β-D-xylopyranoside black colonies will fluoresce,thereby allowing reduction in false positive count of E. coli. XIV8-hydroxy-quinoline-β-D-glucuronide X 6-chloro-3-indolyl-caprylate Xcolor

Black Red-pink XV 8-hydroxy-quinoline-β-D-glucuronide X5-bromo-4-chloro-3-indolyl-caprylate X6-chloro-3-indolyl-α-D-galactopyranoside X X X color

Black Red-pink Blue-violet XVI 8-hydroxy-quinoline-β-D-glucuronide X5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside X X Inhibitor (or)present 5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside X X color

Black Teal (or) Magenta XVII 5-bromo-4-chloro-3-indolyl-β-D-glucuronideX 6-chloro-3-indolyl-β-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X x color

Dark Light Blue- Pink Teal Purple/ Gray Blue XVIII5-iodo-3-indolyl-β-D-glucuronide X6-chloro-3-indolyl-β-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X color

Dark Light Blue- Pink Teal Purple Gray XIX indoxyl-β-D-glucuronide X6-chloro-3-indolyl-β-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X color

Dark Light Blue- Pink Teal Blue/ Gray Purple XX4-methylumbelliferyl-β-D-glucuronide X6-chloro-3-indolyl-β-D-galactopyranoside X X X5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside X X X color

Fluoresce Light Blue- Pink Teal under UV Gray XXI Use same substrates asin example No. XVII-XIX, X X X X and add:4-methyl-umbelliferyl-β-D-glucuronide color

Dark Light Blue- Pink Teal Blue/ Gray Purple and fluoresce

Example I

[0122] The microorganisms chosen to be identified, quantified anddifferentiated are E. coli, general coliforms, Aeromonas and/orSalmonella.

[0123] With reference to Table IV, E. coli produces the enzyme Bgluc,and Bgluc is not produced by any of the other microorganisms desired tobe detected. With reference to the right hand side of Table V, it can beseen that the test enzyme Bgluc has a corresponding substrate componentof β-D-glucuronide. Thus, a chromogenic or nonchromogenic componentwhich produces a distinct color upon cleavage of Bgluc should be chosenfrom the left hand side of Table V. 8-hydroxyquinoline is chosen for itspreferred substantially black color. The first identified substrate istherefore 8-hydroxyquinoline-β-D-glucuronide, the availability of whichis described above. A metallic salt such as ferric ammonium citrate isalso required and is added to the test medium so that, upon cleavage ofthe substrate by Bgluc, a substantially black water-insoluble complexforms in the medium. The substantially black precipitate consists of theferric ions and the aglycone released when the substrate is hydrolyzedby the glucuronidase from E. coli.

[0124] With further reference to Table IV, Bgal, Bfuc and Bglu arecommon to Aeromonas and general coliforms. However, as indicated inTable IV, Bgal, Bfuc and Bglu are not produced generally by Salmonella.Therefore, a substrate component corresponding to one of Bgal, Bfuc andBglu can be selected form the right hand side of Table V. Bgal and theassociated substrate component β-D-galactopyranoside are chosen. The6-chloro-3-indolyl- chromogenic component produces a red-pink color uponcleavage from its substrate in the presence of Bgal and is selected asthe chromogenic component. The second substrate is therefore6-chloro-3-indolyl-β-D-galactopyranoside.

[0125] Again referring to Table IV, Bman, Aara and Agal are common toSalmonella and general coliforms. However, as indicated in Table IV,Bman, Aara and Agal are not produced by Aeromonas. Thus, one of Bman,Aara and Agal can be chosen and its associated substrate componentidentified with reference to Table V. The test enzyme Agal and therespective substrate component α-D-galactopyranoside are chosen. Next, achromogenic component must be selected from Table V. As shown on theleft hand side of Table V, the chromogenic component5-bromo-4-chloro-3-indolyl produces a teal color upon cleavage from itsassociated substrate and is therefore selected. The third substrate istherefore 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside.

[0126] General coliforms have a wide enzyme pattern which is responsiveto both the 6-chloro-3-indolyl-β-D-galactopyranoside substrate and the5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside substrate. Therefore,general coliforms will show as a fourth distinct color which is acombination of the colors produced by the two aforementioned substrates,respectively. In this case the fourth color will be violet-blue, whichis a combination of red-pink and teal.

[0127] Finally, as seen in Table IV, E. coli also exhibits a wide enzymepattern and responsive to all three of the substrates chosen in thisexample, viz., 8-hydroxyquinoline-β-D-glucuronide,6-chloro-3-indolyl-β-D-galactopyranoside, and5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside. Nonetheless, E. colicolonies present in the test medium will show as a substantially blackcolor because, as discussed above, the chromogenic substrates do notinterfere with the substantially black color. Advantageously, thissubstantially black color provides a superior means for distinguishingthe E. coli, as well as allows four separate microorganisms to bedetected, quantified, differentiated and identified in a single testmedium. See Table VI.

Example II

[0128] The selected microorganisms to be detected, quantified,differentiated and identified are E. coli as a first color; generalcoliforms and Salmonella as a second color; and Aeromonas as a thirdcolor.

[0129] With reference to Table IV, E. coli produces the enzyme Bgluc,and Bgluc is not produced by any of the other microorganisms desired tobe detected. With reference to the right hand side of Table V, it can beseen that the test enzyme Bgluc has a corresponding substrate componentof β-D-glucuronide. Thus, a chromogenic or nonchromogenic componentwhich produces a distinct color upon cleavage of Bgluc should be chosenfrom the left hand side of Table V. 8-hydroxyquinoline is chosen for itspreferred substantially black color. The first identified substrate istherefore 8-hydroxyquinoline-β-D-glucuronide, the availability of whichis described above. A metallic salt such as ferric ammonium citrate isalso required and is added to the test medium so that, upon cleavage ofthe substrate by Bgluc, a substantially black water-insoluble complexforms in the medium. The substantially black precipitate consists of theferric ions and the aglycone released when the substrate is hydrolizedby the glucuronidase from E. coli.

[0130] With further reference to Table IV, Bgal, Bfuc and Bglu arecommon to Aeromonas and general coliforms. However, as indicated inTable IV, Bgal, Bfuc and Bglu are not produced by Salmonella. UsingTable V in the fashion described above,6-Chloro-3-indolyl-β-D-galactopyranoside is selected as the secondsubstrate, which will produce a red-pink color upon cleavage asindicated by the chromogenic component list of Table V.

[0131] As seen in Table IV, the enzyme Bman is common to Salmonella butnot Aeromonas. From table V, the substrate component associated withBman is β-D-mannopyranoside. In this example, it is desired to alsoproduce the second distinct color (red-pink) with Salmonella so that,ultimately, Salmonella colonies present in the test medium will show asthe same color as general coliforms present in the test medium. Thus,the chromogenic component is 6-Chloro-3-indolyl- and the third substrateis therefore 6-Chloro-3-indolyl-β-D-mannopyranoside.

[0132] In this example, again using Table V, a fourth substrate isidentified that will be cleaved by one of the enzymes Bman, Aara, Agalcommon to Salmonella to produce a third distinct color. Using table V inthe fashion described above, the fourth substrate selected is5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside, which produces ateal-green color in the presence of Agal common to Salmonella.

[0133] The resulting colors of colonies present in the test medium canbe predicted as follows. E. coli exhibits a wide enzyme pattern that ispositive for all four of the substrates chosen in this example,including the 8-hydroxy-glucuronide substrate which produces asubstantially black color upon cleavage in the presence of the ions ofthe ferric salt. E. coli colonies show as substantially black. Aeromonashas an enzyme pattern which reacts with only the6-Chloro-3-indolyl-β-D-galactopyranoside substrate chosen in thisexample and therefore colonies of Aeromonas show as red-pink. Salmonellahas an enzyme pattern which cleaves both the third and fourth substratesselected in this example and therefore colonies of Salmonella show aspurple-blue (a combination of teal and red-pink). Finally, generalcoliforms are positive for each of the second, third and fourthsubstrates selected and colonies thereof show as purple-blue,indistinguishable from the Salmonella colonies. As discussed above,different strains of all species of the various genera will not allproduce the same amounts of the various enzymes, so there may be slightvariations in shades of purple-blue, for example.

Example IIIA

[0134] The selected microorganisms to be quantified and differentiatedin this example are E. coli as a first color, general coliforms andAeromonas as a second color, and Salmonella as a third color.

[0135] With reference to Table IV, E. coli produces the enzyme Bgluc,and Bgluc is not produced by any of the other microorganisms desired tobe detected. With reference to the right hand side of Table V, it can beseen that the test enzyme Bgluc has a corresponding substrate componentof β-D-glucuronide. Thus, a chromogenic or nonchromogenic componentwhich produces a distinct color upon cleavage of Bgluc should be chosenfrom the left hand side of Table V. 8-hydroxyquinoline is chosen for itspreferred substantially black color. The first identified substrate istherefore 8-hydroxyquinoline-β-D-glucuronide, the availability of whichis described above. A metallic salt such as ferric ammonium citrate isalso required and is added to the test medium so that, upon cleavage ofthe substrate by Bgluc, a substantially black water-insoluble complexforms in the medium. The substantially black precipitate consists of theferric ions and the aglycone released when the substrate is hydrolizedby the glucuronidase from E. coli.

[0136] Using tables IV and V in a fashion similar to that describedabove with reference to Examples I and II,6-Chloro-3-indolyl-β-D-galactopyranoside is selected as a secondsubstrate to combine with one of the enzymes Bgal, Bfuc and Bglu commonto coliforms and Aeromonas, but negative for Salmonella to produce asecond distinct color, in this case substantially red-pink.

[0137] Similarly, 6-Chloro-3-indolyl-α-D-galactopyranoside is selectedas a third substrate to combine with Agal, which is common to coliformsand Salmonella, but negative for Aeromonas. Upon reaction with theenzyme, this substrate will also produce the same distinct second color,namely red-pink.

[0138] 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside is selected as afourth substrate to combine with the enzyme Bgal, which is common tocoliforms and Aeromonas, but negative for Salmonella. This fourthsubstrate produces a teal-green color upon reaction with Bgal.

[0139] The resulting colors of colonies present in the test medium canbe predicted as follows. E. coli exhibits a wide enzyme pattern and ispositive for all four of the substrates chosen in this example.Therefore, E. coli colonies will show as substantially black. Generalcoliform colonies have an enzyme pattern which is positive for thesecond, third and fourth substrates, so that general coliforms coloniesshow as purple-blue. Aeromonas colonies have an enzyme pattern which ispositive for the second and fourth substrates chosen so that Aeromonascolonies also show as purple-blue. Finally, the enzymes common toSalmonella are only positive for the third of the four substrates, sothat Salmonella colonies show as red-pink.

Example IIIB

[0140] As a variation, the test medium of Example IIIA can be preparedsuch that colonies of Salmonella will show as teal instead of pink-red,all of the other colony colors being the same as Example IIIA. Withreference to Table VI, such can be accomplished by replacing the6-chloro-3-indolyl-α-D-galactopyranoside of Example IIIA with5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside.

Example IIIC

[0141] A second, independent method for producing the same three colorsas Example IIIA for the same four components can be achieved by addingnalidixic acid or other antibiotics or inhibitors of Aeromonas to thecomponents listed in Example 1. In so doing, the cefsulodin or nalidixicacid or other substance acts as an inhibitor for Aeromonas so Aeromonascolonies do not grow. If Aeromonas is eliminated, then the purple-bluecolonies are all true coliforms. If not eliminated, any Aeromonas willbe counted as part of the coliforms which some persons may prefer sinceAeromonas is an important indicator organism.

Example IV

[0142] In this example, the selected microorganisms to be detected,quantified and differentiated are E. coli, coliforms and Salmonella as afirst distinct color and Aeromonas as a second distinct color. One testmedium which achieves this result is the test medium described inExample II, except that the first substrate and metallic salt areomitted. Thus, because the enzyme pattern of E. coli reacts with thesame substrates as the enzyme pattern for general coliforms, E. coli andgeneral coliforms will be the same color in this test medium.Specifically, E. coli, coliforms and Salmonella colonies will show as apurple-blue color, whereas Aeromonas colonies will show as asubstantially red-pink color.

Example V

[0143] The selected microorganisms to be detected, quantified anddifferentiated are E. coli, general coliforms and Aeromonas as a firstdistinct color, and Salmonella as a second distinct color. One testmedium which achieves this result is the test medium of Example 3 withthe first substrate and metallic salt being omitted. In this testmedium, E. coli, general coliforms and Aeromonas colonies will show as agenerally purple-blue color, whereas Salmonella colonies will show as agenerally teal-green color or as a red-pink color.

[0144] Optionally, the 6-chloro-3-indolyl-α-D-galactoside can bereplaced with 5-bromo-4-chloro-3-indolyl-β-D-galactoside so thatSalmonella colonies show as teal, rather than pink.

[0145] A third way to achieve the same result is with an antibiotic,preferably nalidixic acid, to inhibit the growth of Aeromonas colonies.If Aeromonas is eliminated, then the purple-blue colonies are all truecoliforms. If not eliminated, any Aeromonas will be counted as part ofthe coliforms which some persons may prefer since Aeromonas is animportant indicator organism.

Example VI

[0146] The selected microorganisms to be detected, quantified anddifferentiated are E. coli and coliforms as a first distinct color,Aeromonas as a second distinct color and Salmonella as a third distinctcolor. A test medium which achieves this result is the test medium ofExample I with the first substrate and metallic salt being omitted. Insuch a test medium, E. coli and general coliform colonies will show aspurple-blue, Aeromonas colonies will show as generally red-pink, andSalmonella colonies will show as generally teal-green.

Example VII

[0147] The selected microorganisms to be detected, quantified anddifferentiated are E. coli as a first distinct color which issubstantially black; general coliforms as a second distinct color whichis substantially purple-blue; Aeromonas/Vibrio/Plesiomonas as a thirddistinct color which is substantially red-pink; and Salmonella as afourth distinct color which is substantially teal-green.

[0148] With reference to Table IV, E. coli produces the enzyme Bgluc,and Bgluc is not produced by any of the other microorganisms desired tobe detected. Therefore, a substrate which produces a distinct color uponcleavage of Bgluc should be chosen from Table V.8-hydroxyquinoline-β-D-glucuronide produces a substantially black colorin the presence of Bgluc and would be the preferred choice of substrate,as explained below. A metallic salt such as ferric ammonium citrate isalso added to form a substantially black water insoluble complexconsisting of the ferric ions and the aglycone released when thesubstrate is hydrolyzed by the glucuronidase from E. coli.

[0149] With further reference to Table IV, it can be seen that theenzyme Ngal and Naglu are common to the microorganisms Aeromonas,Plesiomonas, and Vibrios. Therefore, a suitable substrate for testingall of these microorganisms as a single distinct color is6-chloro-3-indolyl-N-acetyl-β-D-galactosaminide, which produces asubstantially red-pink color in the presence of these enzymes.

[0150] Again referring to Table IV, Bman, Aara and Agal are common toSalmonella and general coliforms. However, as indicated in Table IV,Bman, Aara and Agal are not produced by Aeromonas. Therefore, asubstrate can be selected from Table V which reacts with one of Bman,Aara and Agal to produce a third distinct color. As shown in Table V,5-bromo-4-chloro-3-indolyl-α-D-galactoside produces a teal-green colorin the presence of Agal and is therefore selected as a substrate.

[0151] In this test medium E. coli colonies will show as substantiallyblack, general coliform colonies will show as substantially purple-blue,Aeromonas, Vibrio and Plesiomonas colonies will show as substantiallyred-pink, and Salmonella colonies will show as substantially teal.

Example VIII

[0152] The selected microorganisms to be detected, quantified anddifferentiated are E. coli as a first distinct color; coliforms andSalmonella as a second distinct color; and Aeromonas, Vibrio andPlesiomonas as a third distinct color. One test medium for achievingthis result is the test medium of Example 2, except that the fourthsubstrate chosen is 6-Chloro-3-indolyl-N-acetyl-α-D-galactosaminide, towhich each of the microorganisms Plesiomonas, Vibrios and Aeromonas areresponsive so that each of these colonies shows as a generally red-pinkcolor.

Example IX

[0153] The selected microorganisms to be detected, quantified anddifferentiated in this example are E. coli and general coliforms as afirst distinct color which is purple-blue; Aeromonas, Plesiomonas, andVibrios as a second distinct color which is red-pink; and Salmonella asa third distinct color which is teal-green. This result can be achievedwith the test medium as described in Example 6 with the addition of6-Chloro-3-indolyl-N-acetyl-β-D-galactosaminide, to which each of themicroorganisms Plesiomonas, Vibrio and Aeromonas is responsive.

Example X

[0154] The selected microorganisms to be detected, quantified anddifferentiated in this example are E. coli and general coliforms as afirst color; Aeromonas, Vibrio and Plesiomonas as a second distinctcolor; and Salmonella as a third distinct color. A suitable test mediumthat achieves this result is the test medium disclosed in Example 7except that the first substrate for detecting E. coli colonies isomitted. In this example, E. coli and general coliform colonies show asgenerally purple-blue, Aeromonas, Vibrio and Plesiomonas show asgenerally red-pink, and Salmonella show as generally teal-green. Theaddition of 6-Chloro-3-indolyl-β-D-galactopyranoside is necessary toyield the purple-blue color for E. coli colonies.

Example XI

[0155] The selected microorganisms to be detected, quantified anddifferentiated in this example are E. coli as a substantially blackcolor and general coliforms as a red-pink color. With reference to TableIV, E. coli produces the enzyme Bgluc, and Bgluc is not produced by anyof the other microorganisms desired to be detected. Therefore, asubstrate which produces a distinct color upon cleavage of Bgluc shouldbe chosen from Table V. 8-hydroxyquinoline-β-D-glucuronide produces adark color in the presence of Bgluc and would be the preferred choice ofsubstrate. A metallic salt such as ferric ammonium citrate is also addedto form a black water insoluble complex consisting of ferric ions andthe aglycone released when the substrate is hydrolized by glucuronidasefrom E. coli.

[0156] With further reference to Table IV, Bgal, Bfuc and Bglu arecommon to Aeromonas and general coliforms. However, as indicated inTable IV, Bgal, Bfuc and Bglu are not generally produced by Salmonella.Therefore, a substrate can be selected from Table V which reacts withone of Bgal, Bfuc and Bglu to produce a second distinct color.6-chloro-3-indolyl-β-D-galactopyranoside produces a pink color in thepresence of Bgal and is selected as the second substrate.

[0157] Optionally, the 6-chloro-3-indolyl-β-D-galactopyranoside can bereplaced with 5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside so thatAeromonas and general coliform colonies show as teal instead of pink.

[0158] As noted, the second substrate selected will result in coloniesof Aeromonas also showing as a generally red-pink color. To avoid growthof Aeromonas colonies, an inhibitor, preferably nalidixic acid, isadded. Thus, colonies of E. coli will show as substantially black,whereas colonies of general coliforms will show as a red-pink color.

Example XII

[0159] The selected microorganisms to be detected, quantified anddifferentiated in this example are E. coli, general coliforms andAeromonas spp. as a substantially black color and Salmonella spp. as asecond distinct color. The first substrate selected is8-hydroxyquinoline-β-D-galactoside, which results in colonies of E.coli, general coliforms and Aeromonas showing as substantially black.The second substrate chosen can be either5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside or6-chloro-3-indolyl-α-D-galactopyranoside. If the former of these twosubstrates is chosen, colonies of Salmonella will show as a teal color,whereas if the latter of the two aforementioned substrates is chosen,colonies of Salmonella will show as a red-pink color.

[0160] Optionally, in this example, Aeromonas may be eliminated byadding an inhibitor, preferably nalidixic acid, as discussed in detailabove.

Example XIII

[0161] The selected microorganisms to be detected, quantified anddifferentiated in this example are the same as in Example 1, except thatthis example illustrates a correction for false positives. That is, itis possible that certain unusual Enterobacter and Klebsiella spp. willshow as black colonies along with E. coli in the test medium disclosedin Example 1. Thus, the count of E. coli could be inaccurately high.

[0162] In this example, 4-methyl-umbrelliferyl-β-D-xylopyranoside isadded to the test medium described in Example 1. In so doing,Enterobacter and Klebsiella spp. showing as black colonies will alsofluoresce, thereby allowing reduction in the false positive count of E.coli. This example illustrates the flexibility of embodimentsincorporating the present invention. The fluoroescent component does notinterfere with the substantially black color so that the black coloniesare easily distinguished with the naked eye. Yet, under ultravioletlight, false positives can be detected and substantially reduced byexamining the black colonies for fluorescence.

Example XIV

[0163] The selected microorganisms to be detected, quantified,differentiated and identified are E. coli as a substantially black colorand Salmonella spp. as pink-red. General coliforms are colorless in thisexample.

[0164] With reference to Example I, E.coli is responsive to8-hydroxy-quinoline-β-D-glucuronide. General coliforms, Salmonella andAeromonas are not responsive to 8-hydroxy-quinoline- β-D-glucuronide.Thus, the first substrate chosen is 8-hydroxy-quinoline-β-D-glucuronide.

[0165] With reference to table IV, esterase enzyme is positive forSalmonella spp., but not any of the other preferred microorganisms to bedetected. With reference to table V, the substrate6-chloro-3-indolyl-caprylate can be identified, and will produce apink-red color upon cleavage, and is therefore chosen as the secondsubstrate.

[0166] In this test medium, colonies of E. coli will show assubstantially black and colonies of Salmonella will show as pink-red.

Example XV

[0167] The selected microorganisms to be detected, quantified,differentiated and identified are E. coli as a substantially blackcolor, Salmonellaspp. as dark blue-purple, and general coliforms asred-pink.

[0168] With reference to Example I, E. coli is responsive to8-hydroxy-quinoline-β-D-glucuronide. General coliforms, Salmonella andAeromonas are not responsive to 8-hydroxy-quinoline-β-D-glucuronide.Thus, the first substrate chosen is 8-hydroxy-quinoline-β-D-glucuronide.

[0169] With reference to tables IV and V,5-bromo-4-chloro-3-indolyl-caprylate can be identified as the secondsubstrate to which Salmonella will be responsive. With further referenceto Table V, 5-bromo-4-chloro-3-indolyl-caprylate forms a teal color uponcleavage.

[0170] 6-chloro-3-indolyl-α-D-galactopyranoside, which produces apink-red color upon cleavage, is chosen as the third substrate to whichE.coli general coliforms and Salmonella are responsive.

[0171] In this example, E. coli colonies show as substantially black,general coliform colonies show as red-pink, and Salmonella show asblue-violet (=red-pink+teal).

Example XVI

[0172] The selected microorganisms to be detected, quantified anddifferentiated in this example are E. coli as a substantially blackcolor and general coliforms as a second distinct color.

[0173] With reference to Table IV, E. coli produces the enzyme Bgluc,and Bgluc is not produced by any of the other microorganisms desired tobe detected. Therefore, a substrate which produces a distinct color uponcleavage of Bgluc should be chosen from Table V.8-hydroxyquinoline-β-D-glucuronide produces a dark color in the presenceof Bgluc and would be the preferred choice of substrate. A metallic saltsuch as ferric ammonium citrate is also added to form a black waterinsoluble complex consisting of ferric ions and the aglycone releasedwhen the substrate is hydrolized by glucuronidase from E. coli.

[0174] With further reference to Table IV, Bgal, Bfuc and Bglu arecommon to Aeromonas and general coliforms. However, as indicated inTable IV, Bgal, Bfuc and Bglu are not generally produced by Salmonella.Therefore, a substrate can be selected from Table V which reacts withone of Bgal, Bfuc and Bglu to produce a second distinct color.5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside can be chosen as thesecond substrate, in which event colonies of E. coli will appear assubstantially black and general colifom colonies will appear as teal.Optionally, 5-bromo-6-chloro-3-indolyl-galactopyranoside can be chosenas the second substrate, in which event colonies of E. coli will appearas substantially black and general colifom colonies will appear magenta.To avoid growth of Aeromonas colonies, an inhibitor, preferablynalidixic acid, is added. Thus, colonies of E. coli will show assubstantially black, whereas colonies of general coliforms will show asa magenta color.

Example XVII

[0175] Some selected micro-organisms that can be detected, quantifiedand differentiated in this example are E. coli, general coliforms,Aeromonas, and/or Salmonella. The substrates5-bromo-4-chloro-3-indolyl-β-D-glucuronide,6-chloro-3-indolyl-β-D-galactopyranoside, and5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside are added in quantitiesof approximately 125 mg/l medium; 200 mg/l medium; and 65 mg/l medium,respectively. The remaining preparation and inoculation of the testmedium in this example is similar to that discussed above, except thations of salt are not required when the medium does not have anonchromogenic substrate. In this medium, E.coli, which reacts with allof the substrates, will appear as a very dark blue and/or purple colorbecause the high concentration of the β-D-glucuronide will predominate.The general coliforms, which react to both the α- andβ-D-galactopyranoside appear as a light blue-gray color. Salmonella,which reacts with the α-D-galactopyranoside will have a teal color andAeromonas, which reacts with β-D-galactopyranoside will have a pink-redcolor. It can be beneficial if the concentration/amount used of theβ-D-glucuronide is greater than the β-D-galactopyranoside to increasethe difference in coloration/darkness between the E. coli and generalcoliforms, since these substrates utilize the same color compound inthis example. However, even if the same amounts of β-D-glucuronide andβ-D-galactopyranoside are used, the E. coli may still be darker anddistinguishable from general coliforms since it reacts to both of thesesubstrates whereas the general coliforms do not react with theβ-D-glucuronide.

Example XVIII

[0176] An alternate β-D-glucuronide substrate that may be utilized is5-iodo-3-indolyl-β-D-glucuronide, which is commonly known as Iodo-Gluc.Selected micro-organisms to be detected, quantified and differentiatedin this example are E. coli, general coliforms, Aeromonas, andSalmonella. The concentration of the β-D-glucuronide must be sufficientto provide a very dark purple color that can be readily distinguishedfrom the blue-gray color of the general coliforms.

Example XIX

[0177] Another alternate β-D-glucuronide substrate that may be used isindoxyl-β-D-glucuronide, which is commonly known as IBDG. With asufficient concentration of IBDG, E. coli will appear darker than theother colonies as a dark blue-purple color. General coliforms,Salmonella, and Aeromonas will appear as light blue-gray, teal andred-pink, respectively.

Example XX

[0178] In this example, 4-methylumbelliferyl-β-D-glucuronide, commonlyknown as MUGluc, is used instead of a chromogenic or nonchromogenicβ-D-glucuronide. With this medium, E. coli will fluoresce underultra-violet light, and general coliforms will be light blue-gray,Salmonella will be teal, and Aeromonas will be pink-red in ambientlight. An advantage of the MUGluc is that the incubation times requiredfor detection of the colonies may be substantially less than thatrequired with the other chromogenic or nonchromogenic substrates. Anincubation time of about 14 hours should be sufficient to detect E. coliwith this substrate. A disadvantage is that the fluorescent products aremore readily diffusible than the other compounds and may make it moredifficult to quantify the E. coli. However, even if the E. coli can notbe quantified in a given test, it will still certainly be suitable for apresence/absence test for E. coli.

Example XXI

[0179] In this medium, a 4-methylumbelliferyl-β-D-glucuronide (MUGluc)is combined with one of the other previously mentioned chromogenic ornonchromogenic glucuronide substrates as well as with chromogenic α- andβ-D-galactopyranosides. This medium offers the advantage that apresence/absence test for E. coli may be performed with shorterincubation times than required for the chromogenic and nonchromogenicsubstrates. In addition, if for any reason, it is uncertain whethercolonies of detected organisms are E. coli or general coliforms, themedium can be examined under ultra-violet light so that colonies of E.coli can be confirmed by fluorescence. This provides a double check onthe accuracy of the identity of the colonies.

[0180] Although several broad examples which incorporate the presentinvention have been described above, it is to be understood that thepresent invention is not to be limited by the examples disclosed herein.Indeed, the disclosure and examples above teach one of ordinary skill avirtually limitless number of test media which would be within the scopeof the claims appended hereto.

[0181] Further, while this invention has been described as having apreferred design, the present invention can be further modified withinthe spirit and scope of this disclosure. This application is thereforeintended to cover any variations, uses, or adaptations of the inventionusing its general principles. Further, this application is intended tocover such departures from the present disclosure as come within knownor customary practice in the art to which this invention pertains andwhich fall within the limits of the appended claims.

What is claimed is:
 1. A test medium for detecting, quantifying ordifferentiating general coliforms, E. coli, Aeromonas spp. andSalmonella spp., said test medium comprising: a β-D-glucuronidesubstrate, which forms a first product in the presence of E. coli; anα-D-galactoside chromogenic substrate, which forms a second product inthe presence of the Salmonella; and a β-D-galactoside chromogenicsubstrate, which forms a third product in the presence of Aeromonas,said products of said α-D-galactoside and said β-D-galactosidesubstrates combining to form a color in the presence of generalcoliforms, so that general coliforms, E. coli, Aeromonas, and Salmonellaare distinguishable from one another.
 2. The test medium of claim 1,wherein said β-D-glucuronide substrate and one of either saidα-D-galactoside chromogenic substrate or said β-D-galactosidechromogenic substrate consist of the same color component, but indifferent amounts.
 3. The test medium of claim 2, wherein saidβ-D-glucuronide substrate is 5-bromo-4-chloro-3-indolyl-β-D-glucuronideand said α-D-galactoside substrate is5-bromo-4-chloro-3-indolyl-α-D-galactoside.
 4. The test medium of claim3, wherein said medium has a concentration of approximately 125 mg/l ofβ-D-glucuronide and 65 mg/l of α-D-galactoside.
 5. The test medium ofclaim 1, wherein said β-D-glucuronide substrate is5-iodo-3-indolyl-β-D-glucuronide.
 6. The test medium of claim 1, whereinsaid β-D-glucuronide substrate is indoxyl-β-D-glucuronide.
 7. The testmedium of claim 1, wherein said β-D-glucuronide substrate produces aproduct in the presence of E. coli that fluoresces under ultra-violetlight.
 8. The test medium of claim 7, wherein said β-D-glucuronidesubstrate is 4-methylumbelliferyl-β-D-glucuronide.
 9. The test medium ofclaim 7, further including a second β-D-glucuronide substrate, saidsecond β-D-glucuronide substrate forming a product of color in thepresence of E. coli that is detectable under ambient light anddistinguishable from said other products.
 10. The test medium of claim9, wherein said second β-D-glucuronide substrate is selected from thegroup consisting of 8-hydroxyquinoline-β-D-glucuronide, an esculetinglucuronide, cyclohexenoesculetin-β-D-glucuronide,5-bromo-4-chloro-3-indolyl-β-D-glucuronide,5-iodo-3-indolyl-β-D-glucuronide, and indoxyl-β-D-glucuronide.
 11. Atest medium for detecting, identifying, and qualifying or quantifyingbiological entities, said medium comprising: a nutrient base medium; anda first substrate, which forms a first product in the presence of afirst biological entity; a second substrate, which forms a secondproduct in the presence of a second biological entity; and thirdsubstrate, which forms a third product in the presence of a thirdbiological entity, said first, second, and third products beingdistinguishable from one another, said first product and said secondproduct including the same color forming compound, but in amountssufficiently different to enable said first and second products to bedistinguished from on another.
 12. The test medium of claim 11, whereinsaid first substrate is a β-D-glucuronide substrate, said secondsubstrate is an α-D-galactoside substrate, and said third substrate is aβ-D-galactoside substrate.
 13. The test medium of claim 12, wherein saidβ-D glucuronide and said α-D-galactoside substrates include thecomponent 5-bromo-4-chloro-3-indolyl.
 14. The test medium of claim 12,wherein said β-D glucuronide substrate contains approximately twice theamount of the 5-bromo-4-chloro-3-indolyl component as theα-D-galactoside substrate.
 15. The test medium of claim 14, includingapproximately 125 mg/l of 5-bromo-4-chloro-3-indolyl-β-D-glucuronide and65 mg/l of 5-bromo-4-chloro-3-indolyl- α-D-galactoside.
 16. The testmedium of claim 12, including a fourth substrate that is also a β-Dglucuronide.
 17. The test medium of claim 16, wherein said first productformed in the presence of the first biological entity is visible underambient light, and said fourth substrate produces a product thatfluoresces under an ultra-violet light in the presence of the firstbiological entity.
 18. A test medium for detecting, quantifying, ordifferentiating general coliforms, E. coli, Aeromonas, and Salmonella,said test medium comprising: a nutrient base medium; a first substrate,which forms a first product in the presence of E. coli; a secondsubstrate, which forms a second product in the presence of Salmonella;and a third substrate, which forms a third product of a third color inthe presence of Aeromonas; a fourth substrate, which forms a fourthproduct in the presence of E. coli, all of said products beingdistinguishable from one another; said first, second, and third productsbeing distinguishable under ambient light, and said fourth productfluorescing under an ultra-violet light.
 19. The test medium of claim18, wherein said first and fourth substrates are β-D-glucuronides, saidsecond substrate is an α-D-galactoside, and said third substrate is aβ-D-galactoside.
 20. The test medium of claim 19, wherein said secondand third substrates form said second and third products, respectively,in the presence of general coliforms, said combined second and thirdproducts being distinguishable from said individual products.